Recombinant il 3

For testing drug cytotoxic/cytostatic effects, BCP‐ALL primografts were cocultured with BM‐MSC (Pal et al., 2016). To this end, BM‐MSC were seeded onto 48‐well plate at a density of 2.5–5.0 × 10⁴ cells per well, in DMEM (low glucose) supplemented with 20% FBS (Gibco), 2 mm of l‐glutamine (Sigma‐Aldrich), and 1% penicillin/streptomycin solution (Sigma‐Aldrich). The next day, BCP‐ALL primograft cells were added at a density of 0.5 × 10⁶ cells·mL⁻¹ in SFEM II media (STEMCELL Technologies) supplemented with 20% FBS (Gibco), 20 ng·mL⁻¹ of recombinant IL‐3 (R&D Systems, Minneapolis, MN, USA), 10 ng·mL⁻¹ of recombinant IL‐7 (R&D Systems), and penicillin/streptomycin solution (Sigma‐Aldrich). After 24 h of coculture, cells were treated with auranofin (AUR) or adenanthin (ADE) at indicated concentration ranges, in a final volume of 700 μL. For each drug concentration, samples were run in duplicate. After 5 days, cells in suspension (BCP‐ALL cells) and adherent cells (BCP‐ALL+MSC) were collected and the number of viable cells was assessed in a hemocytometer. The numbers of viable BCP‐ALL cells in control groups after 5‐day‐long coculture exceeded the number of seeded cells, confirming the growth‐supporting role of BM‐MSC. Primograft samples used in drug‐testing experiments are listed in Table S7.

Primary human ALL cells were obtained after informed consent at West Virginia University Cancer Center; Cincinnati Children’s Hospital Medical Center Respiration Core; and Pittsburgh Biospecimen Core under the approved Institutional Review Broad (IRB) protocols: #1310105737; STUDY19030357 and # 2011-3023. Primary human ALL cells were cultured on hTERT-immortalized primary BM mesenchymal stromal cells (MSCs)^{39 ,40 (link)}. MSCs were seeded at a density of 10⁴ cells/cm² in MSC medium 48 h prior to adding to ALL. ALL cells were seeded onto MSC at a density of 2 × 10⁶ cells/ml in SFEM II medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 20% fetal calf serum (GIBCO, Life Technologies), 20 ng/ml recombinant IL-3 (R&D Systems, Abingdon, UK) and 10 ng/ml recombinant IL-7 (R&D Systems, Minneapolis, MN). ALL cells were harvested every 7 days. Non-adherent cells present in supernatant medium were washed with PBS and passed through a 15 μm filter (pluriSelect Life Science, Leipzig, Germany). ALL cells were separated from MSCs by magnetic cell separation using hCD45 microbeads (Miltenyi Biotec, Auburn CA). Viable ALL cells were counted by Trypan blue exclusion, re-suspended in fresh ALL medium, and seeded onto fresh MSC.

Leukopacks were obtained from the Etablissement Français du Sang. Peripheral blood mononuclear cells (PBMCs) were recovered using density gradient centrifugation. The pDCs were isolated using magnetic beads on AutoMacs (Miltenyi Biotech) as previously described (Ka et al., 2014 (link)). Briefly, pDCs were isolated by depletion of non-pDCs that were retained in the column, while unlabeled pDCs with high purity (90%) were collected in the flow-through. Plasmacytoid dendritic cells were then suspended in RPMI 1640, supplemented with 20 mM HEPES, 10% fetal calf serum, 2 mM L-glutamine, 100 U penicillin/ml, 50 μg/ml streptomycin (Life Technologies), and 10 ng/ml recombinant IL-3 (R&D Systems), as described previously (Dental et al., 2012 (link)) and were stimulated with heat-inactivated (100°C for 30 min) C. burnetii organisms (bacterium-to-cell ratio of 50:1), or CpG-A 10 μg/mL (ODN 2216; InvivoGen) for 8 or 24 h.